Retroviruses are small, membrane-enveloped RNA viruses that were first discovered over 80 years ago. They have been extensively studied because of their importance in helping understand eukaryotic gene expression, their role in elucidating cellular growth factors and oncogenes, their role as human pathogens (particularly in AIDS), and their use as tools to genetically alter host cells, especially for experimental and therapeutic purposes. (Retrovirology is reviewed by Varmus, 1984, Science 240: 1427-1435). The retrovirus life cycle involves 1) attachment to a host cell via specific receptors, 2) entry into the host, 3) replication of the genomic RNA via a DNA intermediate which then integrates into the host chromosome, 4) transcription and translation of virion genes, 5) assembly of viral components into virion particles and 6) budding of the particles from the plasma membrane. When the virion particle buds from the cell surface, it becomes membrane-enveloped.
The cell entry step of the retrovirus life cycle only partly determines the host range of a given retrovirus. It has long been established that avian retroviruses can infect and transform mammalian cells, but do not release infectious or non-infectious virus particles [reviewed by Weiss, 1984 in "RNA Tumor Viruses," 2nd ed., Vol. 1 (Weiss, Teich, Varmus and Coffin, eds.) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 209-260]. For avian Rous sarcoma virus (RSV) the block to particle release appears to occur during virion assembly and budding and that block involves the gag gene product, Pr76.sup.gag [Vogt et al. 1982, J. Virol. 44: 725-730].
The gag gene (which encodes five RSV structural proteins) is one of three genes common to all replication-competent retroviruses, the others being pol (which codes for the reverse transcriptase and related functions) and env (which codes for the envelope glycoproteins). RSV is unique in that it also carries an oncogene, src in addition to these three structural genes. The entire nucleotide sequence of RSV is known (Schwarz et al., 1983, Cell 32: 853-869). A large body of genetic evidence, obtained through the characterization of spontaneous mutations, has suggested that gag is the only viral gene needed for budding and particle formation (reviewed in Dickson et al. 1984. in "RNA Tumor Viruses," op. cit., p. 513-648). That is, non-infectious particles can be released from the cells in the absence of reverse transcriptase, envelope glycoproteins, tumor-inducing protein or genomic RNA. It is only when gag is mutated that the ability to form particles is lost.
Pr76.sup.gag is a polyprotein precursor which is synthesized on cytoplasmic ribosomes from an unspliced, proviral transcript that is identical to the viral genome. This polyprotein is subsequently targeted to the plasma membrane (the site of virus assembly) by a mechanism that is not presently understood. Like all type C retroviruses, RSV does not pre-assemble core structures in the cytoplasm, but rather these structures arise concurrently with the envelopment or budding process. The five internal virion proteins that arise through proteolytic processing of Pr76.sup.gag are designated as follows according to their order in the precursor: NH.sub.2 -p19 (the matrix or membrane-associated protein, MA), p2 and p10 (both of unknown function), p27 (the capsid protein, CA), p12 (the nucleocapsid, NC) and p15-COOH (the protease, PR). As is the case for other retroviruses, the processing of Pr67.sup.gag is poorly understood, but it is believed to occur after the arrival of the precursor at the plasma membrane. However, processing itself does not appear to be a prerequisite to the budding process, since RSV mutants have been found that synthesize truncated forms of Pr76.sup.gag which are not cleaved but are released from the cells in the form of particles (Voynow and Coffin, 1985, J. Virol. 55: 79-85). Mammalian retrovirus processing and budding is also independent of mammalian gag precursor cleavage (Crawford et al. 1985. J. Virol. 53: 899-907).
It is not clear how retroviruses target their gag products to the plasma membrane, though it is widely believed that the MA protein plays a critical role. In the case of mammalian retroviruses, almost all encode Gag proteins having a 14-carbon fatty acid, myristate, at the amino-terminus, and this hydrophobic moiety may play a role in membrane interactions during targeting. The myristic acid addition appears to occur co-translationally, and results in an amide bond between the acyl group and the .alpha.-amino group of glycine following removal of the initiation methionine (reviewed by Schultz et al., 1988, Ann. Rev. Cell Biol. 4: 611-647). Elimination of the myristic acid addition site on the Gag protein of Mason-Pfizer monkey virus (M-PMV) by means of site-specific mutagenesis abrogates M-PMV particle release and Gag precursor processing (Rhee et al. 1987, J. Virol. 61:1045-1053); similar results have been found for murine leukemia virus (MuLV; Rein et al. 1986, Proc. Natl. Acad. Sci. USA 83: 7246-7250).
The RSV Gag protein does not have glycine at position 2 and is not myristylated; hence, the failure of Pr76.sup.gag to be targeted, processed and released by budding from mammalian cells might be due to a requirement for myristic acid addition. In accordance with the present invention, it was discovered that the block to RSV Pr76.sup.gag function in mammalian cells is alleviated by the creation of an amino-terminal myristic acid addition site. Myristic acid addition does not adversely affect particle formation in avian cells; in fact it appears to augment particle formation. Furthermore, it was surprisingly found that low, but easily detected, levels of particle formation occur when the wild type (unmodified) Pr76 is expressed at unusually high levels in mammalian cells by the SV40-based expression vectors of the present invention.
It was also discovered in accordance with the present invention that C-terminal deletions of myristylated Pr76.sup.gag result in a protein that is still processed and budded from a mammalian cell as is full-size, myristylated Pr76.sup.gag. Furthermore, the present invention provided the surprising discovery that heterologous gene sequences can be fused to truncated, myristylated Pr76.sup.gag, and the resulting fusion proteins will be processed and budded from a mammalian or avian cell in membrane-enveloped particles similar to immature virions. This process is known as retrovirus-mediated secretion and provides a method for releasing proteins packaged in membrane-enveloped particles, or membrane vesicles, into the culture medium. The particles can be easily and rapidly collected from the medium by centrifugation, and thus provide a convenient means of obtaining recombinant proteins for rapid purification.
In further investigations it has been discovered that three regions of Gag appear to promote budding. This finding allows construction of fusion proteins in accordance with the present invention which have a minimal amount of the Gag protein needed to enable a cell to produce the fusion protein in a membraneous particle. Specifically, it has been discovered that the three regions of Gag needed for budding and particle formation are amino acids 1-8 (the myristylation site), amino acids 84-174 (from MA and the small p2 domain), and amino acids 417-515 (from CA and NC). However, it is possible that some of these residues are not essential for budding and particle formation and thus even smaller regions of Gag may be used in fusion protein constructs.
Retrovirus-based expression systems are known and are reviewed by Varmus. Some of the systems secrete proteins in soluble form into the culture medium. That is, the secretion occurs via the normal intracellular pathway and the secreted proteins are not contained in membrane vesicles or particles, for example, Weighous et al. 1986, Gene 45:121-129. Further gag gene fusions to other retrovirus genes, such as env, pol, onc (oncogenes, e.g. src) are part of the life cycle of all retroviruses. At present none of these gag retrovirus fusions are known to bud (Felsenstein et al. 1988, J. Virol. 62:2179-2182).
Adams et al. 1987, Nature 329: 68-70, describe fusions of foreign proteins to the yeast TYA gene, a retrotransposon gene homologous to the retrovirus gag gene. Yeast retrotransposons (Ty) form virus-like particles (Ty-VLPs) in a manner analogous to virion formation in retroviruses; however, unlike retroviruses, Ty-VLPs are not budded from the cell, but rather accumulate intracellularly. Like retroviruses, Ty-VLPs are membrane-enveloped particles that do not require cleavage of TYA for production of Ty-VLPs. The mechanism of Ty-VLP particle production is not known. Ty-VLPs are not readily purified. Their purification requires lysing the cells and differential centrifugation to separate cellular components from Ty-VLPs.
In contrast, to Ty-VLPs, the present invention is directed to retrovirus gag fusions with heterologous genes whose products are then exported or secreted, from the cell by the viral budding process. In this process the fusion protein continuously accumulates in the culture medium or extracellular space in membraneous particles. Unlike Ty-VLPs, these particles are readily purified; they are also useful for rapid purification of the fusion protein, as immunogens and as drug delivery systems.
The membraneous particles have several advantages for production of fusion proteins. Because of their large size, the particles are easily pelleted upon centrifugation and thus separated from soluble components in the culture medium. Fusion proteins, or any protein, secreted into the culture via the normal intracellular path cannot be rapidly pelleted. Hence, it is more difficult to separate normally secreted proteins (i.e., not in a particle) from those secreted in a membraneous particle. Further, purification of the fusion protein from the membraneous particle is rapid, since the particles have relatively few components. Additionally, the expression system is highly efficient and allows continual production of the membraneous particles since particle production is not toxic to the cells. In the case of RSV gag fusions expressed in mammalian cells, they were found to have a half-time of about 30 minutes for passage through the cell and release in membraneous particles. Finally, the particles are safe to work with, since they lack a full retrovirus genome and are, therefore, non-infectious.